Abstract Genome integrity, which is crucial for cell survival in response to environmental and endogenous stress, depends upon the Base Excision Repair (BER) pathway proteins. BER recognizes and removes damaged or modified bases and apurinic/apymidinic (also called abasic) sites that result from toxins, oxidative stress, and repair processes. BER damage removal and backbone incision is essential to provide the 3' primer terminus for polymerase to replace the damaged base, so polymorphisms in BER proteins are linked to cancer susceptibility, and inhibition of BER proteins is being tested in clinical cancer trials. Yet, the structural biochemistry controlling base damage recognition, pathway initiation, and damage removal remains incompletely understood. We will focus on key BER enzymes that initiate and implement committed steps in BER repair pathways, show specific recognition of damaged base(s), act in removing or processing the DNA damage, and protect the repair intermediate that would itself be toxic or mutagenic to cells. Our Aims address hypotheses on damaged DNA recognition, catalytic mechanism, and pathway coordination for these proteins that control BER pathway initiation: Aim 1, Uracil DNA N-Glycosylase (UDG/UNG) in deaminated base recognition and removal; Aim 2, N-glycosylase/AP-lyases in oxidized base recognition and removal; Aim 3, Repair endonucleases in damaged DNA backbone incision. In each Aim, the experimental strategy will define the determinants for damage recognition and catalytic mechanism through biochemical, mutational, genetic experiments integrated with structural approaches including both small angle X-ray scattering (SAXS) to characterize conformation and assembly in solution and macromolecular X-ray crystallography (MX) to reveal atomic resolution information. This work will leverage the research strengths and facilities of the investigators. The Tainer laboratory builds upon extensive experience in the MX and SAXS analyses of BER and DNA repair proteins. To help address the central challenge of linking proteins to pathways, John Tainer furthermore developed and directs the SIBYLS MX and SAXS beamline at the Advanced Light Source Synchrotron. The Cunningham laboratory has extensive experience with BER biochemistry and with quantitative mutational analyses and genetic complementation tests in E. coli. Strategically chosen collaborators bring complementary protein and technical expertise to aid rigorous analyses. The proposed structural and biochemical research will test unifying hypotheses relevant to base repair enzyme activities and functions in genomic integrity, including our overall proposition that repair pathway progression and coordination is in part an emergent property of the structural chemistry for individual protein:damaged DNA complexes. Overall, this research aims to provide fundamental knowledge of BER complexes relevant to defining their roles in the regulation of genome fidelity and the mechanisms whereby loss of the functions of these coordinated complexes may lead to sensitivity to environmental toxins, to initiation of degenerative disease and cancer, and also to opportunities for therapeutic interventions.